Porous polymeric cellulose prepared via cellulose crosslinking

ABSTRACT

The invention relates to porous polymeric cellulose prepared via cellulose crosslinking. The porous polymeric cellulose can be incorporated into membranes and/or hydrogels. In preferred embodiments, the membranes and/or hydrogels can provide high dynamic binding capacity at high flow rates. Membranes and/or hydrogels comprising the porous polymeric cellulose are particularly suitable for filtration, separation, and/or functionalization media.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. Ser. No.17/248,915, filed Feb. 12, 2021, which application is a continuationapplication of U.S. Ser. No. 16/173,563, filed Oct. 29, 2018, now U.S.Pat. No. 10,919,986 issued Feb. 16, 2021, which application is acontinuation of PCT/US17/30078, filed on Apr. 28, 2017. Theseapplications are related to and claim priority to U.S. provisionalpatent application Ser. No. 62/424,096 filed on Nov. 18, 2016, and U.S.provisional patent application Ser. No. 62/329,778, filed on Apr. 29,2016, all of which are incorporated herein in their entirety, including,without limitation, the specification, claims, and abstract, as well asany figures, tables, or drawings thereof.

GRANT REFERENCE

This invention was made with government support under Grant No.IIP-1329377, awarded by the National Science Foundation. The Governmenthas certain rights in this invention.

FIELD OF THE INVENTION

The invention relates to porous polymeric cellulose prepared viacellulose crosslinking. The porous polymeric cellulose can beincorporated into membranes and/or hydrogels.

BACKGROUND OF THE INVENTION

Cellulose based membranes offer several advantages over other materialsin filtration applications. Cellulose is a readily available, renewablepolymer that is non-toxic and readily modified to add functionality foradvanced separations. Cellulose is currently used in a variety ofmembrane products including air, water, and oil purification andadsorptive filtration applications.

However, there are several challenges that must be overcome to realizethe advantages of cellulose as a filtration media. Cellulose readilyabsorbs water, swelling when wet. Water absorbance/swelling constrictsmembrane pore spaces and leads to reduced permeability. In addition, thevolume of the entire membrane increases. If the base material ismodified with functional groups to act as a chromatography media,further swelling may occur as water interacts with the functional groupsadded to capture proteins and other molecules of interest. Because totalvolume of the material is increased, volumetric binding capacity (amountof target molecule bound per volume of material) decreases compared to anon-swelling material. In extreme cases of functional group addition(e.g. addition of functional tendrils via ATRP), a hydrogel is formedwith very high binding capacity on a mass basis, but suffering from verylow permeability and low volumetric binding capacity due to materialswelling and pore constriction.

When wet, cellulose is also a very mechanically flexible material. Afterfunctionalization, the material becomes even less structured and doesnot retain its shape. This flexibility makes physically working with thematerial difficult. For example, in packing material into a housing inpreparation for flow through filtration, transferring the material mustbe done with great care or the material folds upon itself and must beseparated at risk of damaging the material. The flexible nature ofcellulose also means that material compression may occur during flowthrough filtration. Because of welling, increased pressures must beapplied to obtain a given flowrate compared to non-swelling materials.Over time, pressure may compact the cellulose, reducing pore spaces andincreasing pressure further. The reduced pore spaces also decreaseadsorption kinetics as target molecules can no longer easily accesscertain binding sites through convection and must rely on slowerdiffusion to reach these sites and adsorb to the material. The result isincreasing pressure requirements over multiple runs and very low maximumflowrates 2 MV/min) and/or very deep membrane beds (>1 cm) to achievehigh binding capacity.

Therefore, reducing the swelling of cellulose is advantageous inincreasing permeability and volumetric binding capacity. In addition, ifmaterial stiffness could be increased, the resulting membrane would beeasier to physically work with and would not compress under flow,maintaining low pressure drop over multiple runs and high bindingcapacity at high flowrates and/or smaller bed depths. Thus, an object ofthe invention is to provide porous cellulose compositions with improvedphysical and kinetic properties as a result of reduced swelling. Afurther object of the invention is to provide porous cellulosecompositions with increased material stiffness, kinetics, anddurability. Other objects and advantages of the invention are apparentfrom the detailed description, figures, and claims.

The invention described herein uses crosslinking using multi-functionalcarboxylic acids to modify cellulose containing membrane materials toreduce swelling, increase stiffness and durability (reuseability). In apreferred embodiment, the compositions can provide high binding capacityat high flowrates after the material is further modified for use as achromatography medium.

While multiple embodiments are disclosed, still other embodiments of thepresent invention will become apparent to those skilled in the art fromthe following detailed description, which shows and describesillustrative embodiments of the invention. Accordingly, the drawings anddetailed description are to be regarded as illustrative in nature andnot restrictive.

BRIEF SUMMARY OF THE INVENTION

An advantage of the invention is that the porous cellulosic materialsprovide increased binding capacity to that of existing cellulosicmaterials. It is an advantage of the present invention that membranesand/or hydrogels prepared with the porous polymeric cellulosiccompositions are particularly suitable for filtration, separation,and/or functionalization media. In an aspect of the invention membranesand/or hydrogels of the invention can provide increased binding capacityand operate at higher flow rates.

In a preferred embodiment, the present invention is directed tocompositions comprising a membrane, hydrogel, or combination thereof,which are comprise cellulose, wherein the cellulose is crosslinked, andwherein the composition comprises pores and/or channels. In an aspect ofthe invention, the preferred embodiment can be a hybrid or non-hybridmembrane.

Yet another preferred embodiment of the invention is directed to methodsof preparing a composition comprising adding a composition comprisingcellulose to a crosslinking system; wherein the crosslinking systemcomprises a crosslinking agent and a catalyst; and curing thecomposition thereafter; wherein the composition comprises pores and/orchannels.

While multiple embodiments are disclosed, still other embodiments of thepresent invention will become apparent to those skilled in the art fromthe following detailed description, which shows and describesillustrative embodiments of the invention. Accordingly, the figures anddetailed description are to be regarded as illustrative in nature andnot restrictive.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph comparing the binding capacity on a volume basis of anexemplary Generation 1 cellulosic membrane of the invention (representedby the dashed line) and a traditional commercially available membrane(represented by the solid line).

FIG. 2 is a graph comparing the binding capacity on a mass basis of twoexemplary cellulosic membrane of the invention (Generation 1 andGeneration 2, represented by the dashed and solid lines, respectively)and a traditional commercially available membrane (represented by thedotted line).

FIG. 3 is a graph comparing the binding capacity on a volume basis oftwo exemplary cellulosic membrane of the invention (Generation 1 andgeneration 2, represented by the dashed and solid lines, respectively)and a traditional commercially available membrane (represented by thedotted line).

FIG. 4 is a graph comparing the pressure versus flow on a volume basisof two exemplary membranes of the invention.

FIG. 5 is a graph comparing the binding capacity on a volume basis of anexemplary membrane of the invention and a traditional commerciallyavailable membrane at differing flow rates.

FIG. 6 is a graph comparing the dynamic capacity on a volume basis of anexemplary membrane of the invention (represented by the solid bars) anda traditional commercially available membrane (represented by the dottedbars) at differing flow rates.

FIG. 7 is a graph comparing the dynamic capacity on a mass basis of anexemplary membrane of the invention (represented by the solid bars) anda traditional commercially available membrane (represented by the dottedbars) at differing flow rates.

FIG. 8 is a graph comparing the relative pressure of an exemplarymembrane of the invention (represented by the solid bars) and atraditional commercially available membrane (represented by the dottedbars) at differing flow rates.

Various embodiments of the present invention will be described in detailwith reference to the figures. Reference to various embodiments does notlimit the scope of the invention. Figures represented herein are notlimitations to the various embodiments according to the invention andare presented for exemplary illustration of the invention.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

The present invention relates to porous cellulosic materials. The porouscellulosic materials can be incorporated into membranes that have manyadvantages over existing membranes. For example, membranes comprisingthe porous cellulosic material can have higher binding capacity and canoperate at higher flow rates than existing membranes.

The embodiments of this invention are not limited to particularcellulosic materials, which can vary and are understood by skilledartisans. It is further to be understood that all terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to be limiting in any manner or scope. For example, asused in this specification and the appended claims, the singular forms“a,” “an” and “the” can include plural referents unless the contentclearly indicates otherwise. Further, all units, prefixes, and symbolsmay be denoted in its SI accepted form.

Numeric ranges recited within the specification are inclusive of thenumbers within the defined range. Throughout this disclosure, variousaspects of this invention are presented in a range format. It should beunderstood that the description in range format is merely forconvenience and brevity and should not be construed as an inflexiblelimitation on the scope of the invention. Accordingly, the descriptionof a range should be considered to have specifically disclosed all thepossible sub-ranges and fractions as well as individual numerical valueswithin that range (e.g. 1 to 5 includes 1, 1.5, 2, 2%, 3, 3.80, 4, and5).

So that the present invention may be more readily understood, certainterms are first defined. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which embodiments ofthe invention pertain. Many methods and materials similar, modified, orequivalent to those described herein can be used in the practice of theembodiments of the present invention without undue experimentation, thepreferred materials and methods are described herein. In describing andclaiming the embodiments of the present invention, the followingterminology will be used in accordance with the definitions set outbelow.

The term “about,” as used herein, refers to variation in the numericalquantity that can occur, for example, through typical measuring andliquid handling procedures used for making concentrates or use solutionsin the real world; through inadvertent error in these procedures;through differences in the manufacture, source, or purity of theingredients used to make the compositions or carry out the methods; andthe like. The term “about” also encompasses amounts that differ due todifferent equilibrium conditions for a composition resulting from aparticular initial mixture. Whether or not modified by the term “about”,the claims include equivalents to the quantities.

The term “mass capacity” as used herein refers to the amount of productbound per mass of adsorbent. Capacity for protein adsorption isconsidered “high” if it is above 200 mg of protein/g adsorbent.

The term “volume capacity” as used herein refers to the amount ofproduct bound per dry volume of adsorbent. Capacity for proteinadsorption is considered “high” if it is above 100 mg protein/ml ofadsorbent.

The term “flowrate” as used herein refers to the volume of liquidexpressed in dry membrane volumes (MVs) flowed through membrane per timeexpressed in minutes. Flowrate is considered high if it is above 20MV/min.

The term “composite nanofibers” as used herein are nanofibers producedfrom at least two different polymers.

The term “differentially removable” as used herein means that, when thehybrid nanofiber felt contains at least two different nanofibersprepared with different polymers, conditions can be selected (elevatedtemperature and/or solvent exposure) to remove one of the nanofibers orpolymers forming part of a nanofiber (e.g., in a composite nanofiber).

The term “electrospinning” as used herein refers to the application ofelectric forces to the spin dope to form nanofibers.

The terms “membrane”, “felt”, “mat”, and “screen”, as used herein areinterchangeable and refer to a non-woven, randomly overlaid collectionof fibers, woven collections of fibers, or oriented fibers.

The term “nano fiber felt” as used herein refers to a collection ofnanofibers in a substantially planar array, which may also includemicrofibers added for strength, enhancing flux, etc.

The term “microfibers” as used herein refers to fibers with diameterslarger than 1.0 micrometer, and generally between 1.0 micrometer and 1.0millimeter.

The term “nanofibers” as used herein refers to fibers with diameterssmaller than of 1.0 micrometer, and generally between 10 nanometers and1.0 micrometer, such as between 200 nm and 600 nm.

The term “single component nanofibers” as used herein are nanofibersproduced from at least two different polymers.

The term “spin dope” as used herein refers to the polymer solution thatis used in the electrospinning process.

Crosslinked Cellulose Compositions and Methods of Preparing and Usingthe Same

The compositions of the invention include porous membranes and hydrogelscomprising cellulose. In some embodiments, these compositions can beformed into desired shapes, including, but not limited to fibers,wafers, cylinders, spheres, and hollow tubes. These cellulosiccompositions can be prepared or obtained in prepared form. Thecellulosic composition can be in the form of a hydrogel, membrane,nanofibrous mat, and combinations thereof, and can include nanofibers,microfibers, and mixtures thereof. Preferably the cellulosiccompositions can be porous and/or channeled. The pores and channels canbe nanopores, micropores, nanochannels, microchannels, and combinationsthereof. In certain embodiments, the cellulosic membranes can befunctionalized or have tendrils (preferably Aton Transfer RadicalPolymerization (ATRP) tendrils) or hydrogels attached to their surface.

The cellulosic composition can be a hybrid composition containingcellulose and a non-cellulose-based polymer. Suitable hybrid cellulosiccompositions and methods of preparing the same are described in U.S.Patent Publication No. 2015/0360158, which is incorporated herein in itsentirety. In another embodiment, the cellulosic composition can consistof or consist essentially of cellulose. Such cellulosic compositions,while consisting of or consisting essentially of cellulose in thepolymer structure, can still be surface functionalized and have attachedtendrils or hydrogels.

For example, hybrid cellulosic compositions can be prepared byelectrospinning a hybrid felt with a first non-cellulose-based nanofiberand a composite nanofiber. The first non-cellulose-based nanofiber canbe prepared by electrospinning a first non-cellulose-based polymer;preferably, the first non-cellulose-based nanofiber is a singlecomponent nanofiber. The composite nanofiber can be prepared byelectrospinning a cellulose-based polymer and a secondnon-cellulose-based polymer. In an aspect of the invention, the firstand second non-cellulose-based polymers can be differentially removable.

The hybrid cellulosic compositions can contain at least 30 wt. %, atleast 40 wt. %, at least 50 wt. %, at least 60 wt. %, or at least 70 wt.% cellulosic nanofibers.

Non-Cellulose-Based Polymers

While the majority by mass in a hybrid cellulosic composition can befrom the cellulose-based polymers, incorporation of additional types offibers within the felts can provide functionality desired forapplications of the felts. Accordingly, it is desirable to haveadditional fibers within the felts because they can provide increasedmechanical strength to the felt, allow for multiple functionalities tobe incorporated into the felt, provide stability to the manufacturingprocess, and other aspects as explained elsewhere herein. Indeed, it wasunexpectedly discovered by the present inventors that including even asmall proportion of non-cellulosic-based polymers in the hybridcellulosic compositions improved the electrospinning process and alsoallowed for tailoring of the finished product for a variety ofbiological and industrial applications, especially when the hybridcellulosic compositions contained both a composite nanofiber and asingle component nanofiber.

Synthetic polymer nanofibers (e.g., those produced from vinyl polymersand acrylic polymers) offer a wide range of chemical functionalities forbioseparations and other applications. By combining different polymericunits, the surface chemistry of the resulting fiber can be controlled aspart of the electrospinning process, providing direct functionality tothe produced nanofiber. As an alternative, and similar to conventionalmicrometer scale fibers, the surface functionality of polymer nanofiberscan be chemically modified post-electrospinning to accommodate specificfunctionality requirements for various bioseparation applications.Functionalization chemistries are well known in the polymer arts. Theyalso generally withstand harsh cleaning regimens associated withbioprocesses. Exemplary functionalization chemistries are also discussedin more detail elsewhere herein.

Synthetic carbon-based adsorptive media and filtration membranes areoften much more chemically robust than cellulose-based media, and thuscan be used when strong acids and bases are required for cleaning theseparation media between uses. Furthermore, hybrid nanofibers thatinclude both cellulose-based and non-cellulose-based polymers (e.g.,polyacrylonitrile and polyvinyl alcohol) exhibit even higher specificsurface area and greater mechanical strength when compared to singlecomponent cellulose or single component synthetic polymer nanofibers.Accordingly, there is an observable synergy when composite nanofibersinclude both cellulose and non-cellulose-based polymers.

Many polymers have been successfully electrospun into nanofibers,including (1) thermoplastic homopolymers such as vinyl polymers, acrylicpolymers, polyamides, polyesters, polyethers, and polycarbonates, (2)thermoplastic copolymers such as vinyl-co-vinyl polymers,acrylic-co-acrylic copolymers and vinyl-coacrylic polymers, (3)elastomeric polymers such as triblock copolymer elastomers, polyurethaneelastomers, and ethylene-propylene-diene-elastomers, (4) highperformance polymers such as polyimides and aromatic polyamides, (5)liquid crystalline polymers such as poly(p-phenylene terephthalamide)and polyaramid, (6) textile polymers such as polyethylene terephthalateand polyacrylonitrile, (7) electrically conductive polymers such aspolyaniline, as well as (8) biocompatible polymers (i.e. “biopolymers”)like polycaprolactone, polylactide, chitosan and polyglycolide. Asdescribed, the polymer may also be a copolymer of two or more of theabove-named polymer species.

Examples of the additional polymers that can be added into the hybridnanofiber felts are electrospun as single component nanofibers frompolyacrylonitrile (PAN), polyimides, polyamides (nylon 6, nylon 6,6,nylon 6,10, etc.), polyesters (polyethylene terephthalate, etc.), aswell as copolymers thereof.

Preparation of Cellulosic Compositions

Preferably, production of cellulosic compositions relies on thecellulose acetate form of the molecule due to high solubility incommonly used solvents. After production, cellulose can be obtained byregenerating the material with sodium hydroxide in a solvent containingwater and ethanol. Specifically, a concentration in the range of about0.05 to about 0.5 N NaOH in a regeneration solvent consisting of about 0to about 20 (vol %) ethanol in water is preferred with a regenerationtime of up to about 48 hours. The material can be washed with severalvolumes of water, preferably reverse osmosis (RO) water, and dried atroom temperature. Other methods of preparing cellulosic felts can alsobe employed.

Once a cellulosic composition is obtained, the material can be added toa crosslinking system. The crosslinking system can comprise acrosslinking agent and a catalyst in solution. Preferably thecrosslinking agent and catalyst are in a solution with water.Preferably, the water is RO water.

Suitable concentration ranges of the crosslinking agent can be fromabout 5 to about 100 g/L and catalyst from about 5 to about 100 g/L.Preferred crosslinking agents can include, but are not limited to,aldehydes, organochlorides, ethers, multi-functional carboxylic acids,urea derivatives, glycidyl ethers, and mixtures thereof.

The catalyst used can depend on the crosslinking agent. Preferredcatalysts include, but are not limited to, cyanamide, boric acid,aluminum sulfate, ammonium persulfate, sodium hypophosphite, magnesiumchloride, phosphate-containing compounds, and mixtures thereof.

Preferred crosslinking agents include, but are not limited to, compoundscontaining a multi-functional carboxylic acid and/or catalysts describedabove. Preferred crosslinking agents include, but are not limited to,citric acid, malic acid, maleic acid, itaconic acid-maleic acid, 1,2,3,4buthanetetracarboxylic acid (BTCA), sodium hypophosphite, ammoniumpersulfate, sodium hydroxide, aluminum sulfate, glyoxal, glycerol, 1,4butanediol diglycidyl ether, and mixtures thereof. In some embodiments1,2,3,4 buthanetetracarboxylic acid (BTCA), ammonium persulfate,glycerol, glyoxal, sodium hypophosphite, and combinations thereof aremost preferred crosslinking agents.

The cellulosic composition can be soaked from about 2 minutes to about 1hour in the crosslinking system and removed. In some embodiments, thecrosslinking system can be heated and/or stirred while the cellulosiccomposition is soaking. A portion of the crosslinking solution may beremoved from the cellulosic felt via pressing to obtain a certain amountof crosslinking agent per mole of cellulose andydroglucose units (AGUs).The wet cellulose containing material can then dried. Preferably thedrying is performed at a temperature between about 20° C. and about 100°C. for a time greater than 0 minutes and less than about 120 minutes;most preferably the drying is performed at about 25° C. for betweenabout 30 and about 60 minutes. The drying and curing steps can beperformed separately or simultaneously. In a preferred embodiment, theyare performed separately.

The cellulosic composition is also cured. Preferably, the cellulosecontaining material is cured at a temperature between about 120° C. andabout 195° C. for between about 1 and about 30 minutes. The cellulosecontaining material can be cooled after being at a temperature elevatedabove room temperature. After cooling to room temperature, the materialcan be washed and then dried at room temperature. Preferably thecomposition is washed in water, more preferably in RO water.

In an aspect of the invention cellulosic composition can be layered. Thelayered compositions can have any number of layers, including forexample, between about 2 layers and about 1000 layers.

The cellulosic compositions that have been crosslinked can exhibitsignificantly improved properties compared to the base material. Forexample, the compositions can have increased stiffness, reducedshedding, improved permeability when wet, durability, reuseabilityand/or reduced swelling. The crosslinked cellulosic compositions can bestiffer, hold its shape better, and can be readily packed intofiltration holders. Permeability is not adversely affected by thecrosslinking and with certain crosslinking conditions may be improved.After crosslinking, the compositions can be used directly in filtrationapplications or further modified. The material can further modified toadd functional groups to create other chromatography media (e.g.,cation/anion exchange, affinity, etc.). An additional benefit is thatthe modified cellulose material can contain some unreacted carboxylgroups after the crosslinking. Beneficially, these unreacted carboxylgroups can act as cation exchange groups by themselves withoutadditional modification.

Surprisingly, it was found that the improved permeability present in thecrosslinked cellulose compositions was retained even after furthermodification. This allows for much higher flowrates to be used withlittle loss in binding capacity. Even more surprising was that operatingflowrates in excess of 100 MV/min could be used without significant lossof binding capacity, where the non-stabilized functionalized materialwas limited to <5 MV/min. This may indicate that the improvements inmaterial stiffness translated to a more structured material in whichgood flow distribution and minimal compression allowing for highpermeability and very fast adsorption kinetics.

The crosslinked cellulose compositions can be formed into desiredshapes. In some embodiments, the crosslinked cellulose compositions canbe used as a filtration, separation, and/or functionalization media. Ina preferred embodiment the filtration media is for adsorption andchromatography, including, but not limited to liquid chromatography,HPLC, UPLC, and/or gas chromatography. The cellulosic compositions canbe used to separate many materials, including, but not limited to,biomaterials, microorganisms, proteins, DNA, etc. Preferably, thecellulosic compositions can be used to separate microorganisms,including, but not limited to, viruses, bacteria, yeast and mammaliancells.

Surface Functionalization

After the preparation of a hybrid composition, the fiber surfaces may befunctionalized. Non-limiting examples of functionalization include theaddition of ion-exchange groups such as weak or strong acids, and bases(e.g., carboxylic acids and amines), hydrophobic groups such as phenoliccompounds, and affinity ligands such as virus conjugates, antibodies,enzyme substrates, and small molecule biomimetics.

For use in bioseparation, the hybrid compositions of the presentinvention are ideally biologically inert, meaning that they shouldresist non-specific binding of insoluble solids such as cells andcellular debris, as well as unwanted interactions with proteins, sugars,nucleic acids, viruses, and other soluble components present in manybiologically produced systems.

In addition, nanofiber felts for use in bioseparation should exhibitseveral qualities: (1) small diameter fibers to allow for the largestamount of specific area (this criterion is most important for adsorptionprocesses and less important for strictly size-based separationsdiscussed below); (2) well-controlled and narrow pore size distributionbetween fibers to allow for even flow distribution during adsorptiveapplications and for a tight size cutoff for size-based separations; (3)fibers should have excellent mechanical and chemical stability towithstand potentially high operating pressures and harsh cleaningconditions; and (4) fibers should have a well-defined and spatiallyconsistent size and chemical composition.

For adsorption processes, where macromolecular products such asproteins, nucleic acids, and viruses are the predominant targets, thelarge specific surface area associated with nanofiber felts provides aplurality of potential binding sites for adsorptive bioseparations.Nanofibers can be modified to contain a plurality of binding sites andadsorption can occur on the surface of the fibers, which makes thebinding sites immediately available without requiring the targetmolecule to diffuse internally. Internal diffusion can often limit thecapacity for many adsorption processes of bioproducts when usingtraditional porous resin beads because of the relatively large size ofthe target molecules. In addition, because the nanofiber membranes canbe made from many different polymer and cellulose-based nanofibers, theadsorption ligand can be tailored to meet the needs of a particularseparation (e.g., ionic, hydrophobic, and affinity). In some cases theligand can be incorporated into the nanofiber from the source materialsduring electrospinning, or alternatively the surface can be chemicallymodified to provide the desired adsorbing agent after producing thenanofiber.

Two of the most important characteristics of the separation operationare that, (1) flow is through micro- and macro-pores of the felt (asopposed to tightly packed resin beads), and (2) that adsorption takesplace on the surface of the fibers, where no internal diffusion isrequired. These factors reduce concerns of high-pressure drops withelevated flow rates, and eliminate the slow intra-particle diffusionrequired for adsorption within resin beads. It has been shown that thebinding capacity of biomolecules to currently available adsorptive feltsis similar in magnitude to resin beads, but can operate at processingflow rates over 10 times faster than packed beds. These factors allowfor much faster processing times and potentially higher binding levelsfor purifying valuable biological products. This is highly desirable,especially for large biomolecules (molecular weights greater than 100kDa, and/or hydrodynamic diameters of 20-300 nm), because they aredifficult to purify using packed beds due to the mass transferlimitations within the small pores of resin beads.

The surface of the nanofiber felts of the present invention can bemodified to provide ion-exchange and hydrophobic interaction chemistry.Simple chemical modification such as sulfonation of polystyrene fiberswith sulfuric acid can be used to produce a cation exchange medium.Grafting, atom transfer radical polymerization (ATRP), and plasmatreatments can be used to create ion-exchange surface functional groupsas well as three-dimensional tethers from a variety of polymericsubstrates including polypropylene, polyvinylidene difluoride,polysulphone, and others. Phenyl and butyl groups can also be introducedas hydrophobic interaction ligands. It may be desirable to furthermodify the surface of polymer membranes to increase the hydrophilicityand to discourage non-specific binding. This has been accomplished byintroduction of poly(ethylene glycol) and other polyols onto thesurface.

The ion exchange capacity of a hybrid compositions can also be enhancedby introducing, including for example, but not limited to,diethylaminoethyl (DEAE) groups as a weak anion exchange ligand orcarboxylic acid as a weak cation exchange ligand.

Surface Functionalization with Antimicrobials

In one embodiment of the present invention, the non-cellulose-basedpolymer is polyacrylonitrile (PAN). Fibrous membranes of PAN arepreferable for filtration due to thermal stability, high mechanicalproperties, and chemical resistivity. Electrospun PAN nanofiber feltshave been of particular interest due to properties such as small fiberdiameters and the concomitant large specific surface areas, as well ascapabilities to control pore sizes among nanofibers and to incorporateantimicrobial agents at nanoscale. Felts comprised of nanofibers withantimicrobial functionality have attracted growing attentions due to theconcerns about qualities of purified water and/or filtered air as wellas the processing costs. Water and air filters (particularly thoseoperating in the dark and damp conditions) are constantly subject toattacks from environmental microorganisms. The microorganisms (such asbacteria) that can be readily captured by the filters grow rapidly,resulting in the formation of biofilms. Consequently, the buildups ofmicroorganisms on the filter surfaces deteriorate the qualities ofpurified water and/or filtered air; additionally, they also have theunfavorable effects on the flow of water and/or air.

Moreover, the contaminated filters with biofilms are difficult to clean.Usually, high pressure is required during the operation. This in turnincreases the costs. Reported methods incorporate antimicrobial agents(such as N-halamine and silver ions/nanoparticles) directly into spindopes, thus the molecules/particles of antimicrobial agents aredistributed throughout the nanofibers (Xinbo Sun, Lifeng Zhang,Zhengbing Cao, Ying Deng, Li Liu, Hao Fong, and Yuyu Sun. “ElectrospunComposite Nanofiber Fabrics Containing Uniformly Dispersed AntimicrobialAgents as an Innovative Type of Polymeric Materials with SuperiorAnti-Infective Efficacy”. ACS Applied Materials and Interfaces, 2(4),952-956, 2010.)

However, this often leads to process problems, primarily because thehigh content of antimicrobial agents can seriously affect the process ofelectrospinning and/or deteriorate the properties of the resultingnanofibers. A potential solution to these problems is to introduceantimicrobial functionality onto nanofiber surfaces after the nanofibersare produced (Lifeng Zhang, Jie Luo, Todd J. Menkhaus, HemanthramVaradaraju, Yuyu Sun, and Hao Fong. “Antimicrobial Nano-fibrousMembranes Developed from Electrospun Polyacrylonitrile Nanofibers”.Journal of Membrane Science, 369, 499-505, 2011.)

It is known that the nitrile (—C═N) groups in PAN can be chemicallyconverted into amidoxime (—C(NH₂)═NOH) groups. The amidoxime groups cancoordinate with a wide range of metal ions including silver ions, andthe coordinated silver ions can be reduced into silver nanoparticles.Both silver ions and silver nanoparticles are antimicrobial agents withhigh antimicrobial efficacy.

Other Examples

A promising alternative to packed bed chromatography and otherseparation technologies is the use of the hybrid compositions of thepresent invention as selective adsorptive membranes. This style ofadsorption utilizes the nanofiber felts as the support for ligands thatare used during the selective adsorption process.

Selective adsorption involves “active” surface functionalization of thehybrid nanofiber felt, which allows for direct capture (adsorption) oftarget substances. Such modification is simplified if the hybridcompositions include chemical moieties on their surfaces that arerelatively simple to chemically modify to provide adsorption sites.

Unlike modifying nanofiber surfaces for ion-exchange and hydrophobicinteraction functionality, incorporating affinity ligands onto thenanofiber can be more challenging. Often, the process requires firstmodifying the surface to create coupling sites for immobilization of theligand, followed by attachment of the ligand to the active site.Importantly, both the initial surface modification and the coupling ofligand should be robust as not to leach during processing.

In some cases, simple carboxyl groups from grafting methacrylic acidonto the surface can act as the active coupling site by creating acovalent amide bond between the functionalized carboxyl group and anexposed amine group on a protein ligand. Similarly, strong oxidation ofcellulose (if controlled properly) can provide aldehyde groups on thefiber surface that can form a covalent attachment to primary amines of aprotein (including Protein A and Protein G); especially through theamino acid lysine. In other cases, surface functionalization with ageneral affinity dye (e.g., Cibacron Blue, capable of binding someproteins) can be coupled directly to a cellulose nanofiber.

More elaborately, bio-active sites for protein ligand immobilization canbe incorporated into the nanofiber backbone during nanofeltconstruction. One example of this is using poly ethylene glycol (PEG)with poly D,L lactide (PDLLA) as a block copolymer. The glycol can becoupled with biocytin (capable of affinity interaction with streptavidinfusion proteins) after electrospinning to create an affinity nanofiber.Similarly, a polycaprolactone (PCL) and poly(D,L-lactic-co-glycolicacid)-b-PEG-NH2 (PLGA-b-PEF-NH2) diblock copolymer can be createdcontaining surface aminated nanofibers for coupling with proteins usinga homobifunctional coupling agent. Finally, in some cases it is possibleto use intrinsic active sites associated with certain nanofibermatrices. For instance, coupling Concanavalin A (an affinity tag forlectin associated with glycol-proteins and/or other glycolconjugates) toa chitosan-based nanofiber has been successful.

Other techniques for attaching specific ligands to cellulose-basedcompounds and/or synthetic polymers are known in the chemical arts.

Methods of Using the Composition

In an aspect of the invention, the compositions can be used by flowing afluid through the compositions. Preferably the porous cellulosiccompositions are a membrane and/or hydrogel in such a method. Morepreferably, the porous cellulosic compositions can be incorporated intoa variety a separation, filtration, and/or functionalization media. Themethod can further comprise a step of separating molecules from thefluid. The method can further comprise the step of functionalizing amolecule. In an aspect of the invention, the flow rate of the fluid canbe between 5 MV/min and about 400 MV/min, preferably between about 10MV/min and about 300 MV/min. In an aspect of the invention, thecompositions can have a dynamic binding capacity on a volume basis of atleast about 60 mg/ml of the composition, preferably between about 80mg/ml and about 300 mg/ml. In an aspect of the invention, thecompositions can have a dynamic binding capacity on a mass basis of atleast about 120 mg/g of the composition, preferably between about 150mg/g and about 650 mg/g.

EXAMPLES

Embodiments of the present invention are further defined in thefollowing non-limiting Examples. It should be understood that theseExamples, while indicating certain embodiments of the invention, aregiven by way of illustration only. From the above discussion and theseExamples, one skilled in the art can ascertain the essentialcharacteristics of this invention, and without departing from the spiritand scope thereof, can make various changes and modifications of theembodiments of the invention to adapt it to various usages andconditions. Thus, various modifications of the embodiments of theinvention, in addition to those shown and described herein, will beapparent to those skilled in the art from the foregoing description.Such modifications are also intended to fall within the scope of theappended claims.

Example 1 Stabilization of Cellulose Nanofibers Via Crosslinking withBTCA

Cellulose nanofiber containing materials are stabilized by crosslinkingcellulose with 1,2,3,4 Buthanetetracarboxylic Acid (BTCA) and SodiumHypophosphite in water. A 1.55 g sheet of 85% cellulose, 15% PAN wasimmersed in 20 ml water containing 1.26 g BTCA and 1.30 g sodiumhypophosphite. After soaking for 30 mins, the sheet was removed, placedon aluminum foil, and heat treated. The sheet was first pre-dried at 85C for 15 minutes, followed by a temperature increase to 180 C. Next, thesheet was allowed to cool to room temperature, then immersed in reverseosmosis (RO) water to separate the sheet from the foil backing. Thesheet was then washed with several volumes of RO water to removeresidual BTCA and sodium hypophosphite. The produced sheet exhibitedvisibly enhanced material stiffness and was easier to handle.Permeability is equivalent or less than non-stabilized cellulosenanofiber sheets.

Example 2 Cation Exchange Functionalization of Stabilized CelluloseNanofibers

Cellulose nanofibers stabilized via crosslinking may be modified withcation exchange groups using a two-step surface treatment reaction withsodium hydroxide and chloroacetic acid. The crosslinked material isimmersed in a reaction solution containing from 0-100% 2-propanol inwater along with 1-3 g/L NaOH. After mixing at 20-55 C, chloroaceticacid is added in amounts of 1-5 g/L. The reaction is allowed to proceedfor 1-7.5 hours at 35-60 C. Reaction is terminated by washing thecarboxymethylated cellulose material in several volumes of RO waterfollowed by washing with hot (50-60 C) water for 30 minutes-2 hours. Thematerials obtained from the reaction act as weak cation exchangechromatography media, exhibiting high binding capacity to positivelycharged molecules. The reacted materials retain stiffness and lowswelling behavior observed in the base materials and exhibit highpermeability compared to non-crosslinked cation exchange cellulosematerials. The materials also exhibit high binding capacity on avolumetric basis (>220 mg/ml lysozyme), due to low swelling behavior.The materials may be operated at very high flowrates (>100 membranevolumes per minute) with little loss in capacity.

Example 3 Anion Exchange Functionalization of Stabilized CelluloseNanofibers

Cellulose nanofibers stabilized via crosslinking may also be modifiedwith anion exchange groups using a separate two-step surface treatmentreaction with 2-(diethylamino) ethyl chloride hydrochloride (DAECH) andsodium hydroxide. The crosslinked or non-crosslinked cellulose materialis immersed in a solution of 20-60 g/l NaOH in water. After mixing at50-70 C for 30-60 minutes, a 20% solution of DEAECH in water is added tobring the final DEAECH concentration to between 40-80 g/l. The solutionis then mixed at 50-70 C for an additional 2-5 hours. The reaction isterminated by removing the material from the reaction mixture andwashing with several volumes of room temperature RO water, followed by ahot wash (50-60 C) in water for 30 minutes-2 hours. The producedmaterial acts as an anion exchange chromatography material and exhibitshigh volumetric binding capacity to negatively charged molecules (e.g.bovine serum albumin). If produced from non-stabilized cellulose, theproduced material may be stabilized by heat treatment in the presence ofa crosslinking agent, preferably ammonium persulfate. The producedmaterial is soaked in a solution of 0.1-2 g/l ammonium persulfate inwater for 30-120 minutes. Next, the material is cured at 100-150 C for10-40 minutes.

Example 4 Comparative Testing of Cellulosic Membranes

A cellulosic membrane was prepared according to the methods of theinvention except that it was not crosslinked. This exemplary membrane isreferred to as Generation 1 in these examples. The Generation 1 membranewas tested against commercially available cellulosic membranes (referredto herein as “Commercial Membrane”). The binding capacity on a volumebasis was assessed. The results are shown in FIG. 1 with the Generation1 membrane represented by the dashed line and the existing commercialmembrane represented by the solid line. As can be seen in FIG. 1 , theexemplary Generation 1 membrane had higher adsorption capacity on avolume basis compared to the commercially available cellulose membrane.

A crosslinked cellulosic membrane exemplary of a preferred embodiment ofthe invention was prepared according to the methods of the invention. Itis referred to as the Generation 2 membrane. The Generation 2 membranewas tested against an existing commercially available cellulosicmembrane, as well as Generation 1 membrane. The Generation 1 andGeneration 2 membranes exemplary of preferred embodiments of theinvention were tested for binding capacity on a mass basis in comparisonwith a commercially available cellulose membrane. The results are shownin FIG. 2 where the Generation 1 membrane is represented by the dashedline, the Generation 2 membrane is represented by the solid line, andthe commercially available cellulose membrane is represented by thedotted line. FIG. 2 demonstrates that the exemplary membranes of theinvention provide much higher dynamic capacity at much higher flowrates. The Generation 2 membrane provided the highest dynamic capacitywith about 560 mg/g at 17 MV/min (3.5 second residence time) and about480 mg/g at 87 MV/min (0.7 second residence time), whereas thecommercial cellulose membrane provided less than 100 mg/g at a flow ratebetween 20 MV/min and 80 MV/min.

The Generation 1 and Generation 2 membranes exemplary of preferredembodiments of the invention were also tested for binding capacity on avolume basis in comparison with a commercially available cellulosemembrane. The results are shown in FIG. 3 where the Generation 1membrane is represented by the dashed line, the Generation 2 membrane isrepresented by the solid line, and the commercially available cellulosemembrane is represented by the dotted line. FIG. 3 shows that theexemplary membranes of the invention provide much higher dynamiccapacity at much higher flow rates than the commercially availablecellulose membrane. The exemplary membranes of the invention providedabout 224 mg/mL at 17 MV/min (3.5 second residence time) and about 190mg/mL at 87 MV/min (0.7 second residence time), whereas the commercialcellulose membrane provided about 30 mg/g at a flow rate between 20MV/min and 80 MV/min.

The Generation 1 and Generation 2 membranes of the invention werecompared for pressure versus flow. The results are provided in FIG. 4where the generation 1 membrane is represented by the dashed line andthe generation 2 membrane is represented by the solid line. Thegeneration 1 membrane provided had a bed height of 0.6 mm and theeight-layer membrane had a bed height of 0.8 mm. As can be seen in FIG.4 the exemplary generation 2 membrane of the invention provide high flowrates at low pressures.

As demonstrated in FIGS. 2-4 , the Generation 2 membrane (which wascrosslinked) outperformed the commercially available membrane and theGeneration 1 membrane (not crosslinked). Without wishing to be bound bythe theory it is believed that the crosslinking resulted in improvedproperties higher capacity and lower pressure, enabling higher flowrateoperation.

The Generation 2 exemplary membrane of the invention was tested incomparison with a commercially available cellulosic membrane forlysozyme binding capacity at varying high flow rates with residencetimes below 1 second. The results are shown in FIG. 5 . FIG. 5 shows thebinding capacity of an “Exemplary Membrane” is substantially higher thanthe commercial material even for very high flow rates corresponding tovery short residence times. The data reflected in FIG. 5 is alsoprovided in Table 2 below.

TABLE 2 Flowrate [MV/min] 70 90 110 130 Commercial Product Capacity[mg/ml] 25.6 25.6 25.6 25.6 Exemplary Membrane Capacity [mg/ml] 199 190181 172 Residence time [seconds] 0.86 0.67 0.55 0.46

Example 5 Comparison of Cellulosic Exchange Membranes in Capsules

Two equal volume cellulose-based exchange membranes were tested inpre-packaged capsules. One was an exemplary eight-layer membrane of theand the other was a commercially available cellulosic membrane. Astandard protein absorption-elution sequence was performed by the stepsof: equilibration, loading, washing, eluting, and regeneration. Resultsfrom the data are provided in FIGS. 7-9 where the exemplary membrane ofthe invention is represented by the solid bars and the commerciallyavailable membrane is represented by the dotted bars.

FIG. 6 shows the dynamic volume capacity (at different flow rates of thetwo cellulose-based cation exchange membranes. It is clear from FIG. 6that the exemplary membrane of the invention significantly outperformedthe commercially available cellulose-based exchanged membrane at allflow rates.

FIG. 7 shows the dynamic capacity (dry mass) at different flow rates ofthe two cellulose-based cation exchange membranes. Again, it is clear inFIG. 7 that the exemplary membrane of the invention significantlyoutperformed the commercially available cellulose-based exchangedmembrane at all flow rates.

FIG. 8 shows the relative pressure compared at different flow rates ofthe two cellulose-based cation exchange membranes. Again, the exemplarymembrane of the invention outperformed the commercially availablemembrane.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the inventions and all suchmodifications are intended to be included within the scope of thefollowing claims. The above specification provides a description of themanufacture and use of the disclosed compositions and methods. Sincemany embodiments can be made without departing from the spirit and scopeof the invention, the invention resides in the claims.

1-20. (canceled)
 21. A separation media comprising: a membranecomprising crosslinked cellulose; wherein the membrane comprises aplurality of pores and/or channels; wherein the membrane comprises asurface functionalization comprising a cation exchange group, an anionexchange group, or a ligand; and wherein the membrane comprises at least30 wt. % of the cellulose.
 22. The separation media of claim 21, whereinthe membrane comprises at least 40 wt. % of the cellulose.
 23. Theseparation media of claim 21, wherein the membrane is a woven mat or anon-woven mat.
 24. The separation media of claim 21, wherein the poresand channels comprise micropores, nanopores, microchannels,nanochannels, or a mixture thereof.
 25. The separation media of claim21, wherein the surface functionalization is an attached tendril. 26.The separation media of claim 21, wherein the anion exchange groupcomprises a diethylaminoethyl (DEAE) group.
 27. The separation media ofclaim 21, wherein the cation exchange group comprises a carboxylic acid.28. The separation media of claim 21, wherein the ligand comprises aprotein ligand.
 29. The separation media of claim 21, wherein themembrane further comprises a crosslinking system.
 30. The separationmedia of claim 29, wherein the crosslinking system comprises one or moreof an aldehyde, an organochloride, an ether, a multi-functionalcarboxylic acid, a urea derivative, a glycidyl ether, citric acid, malicacid, maleic acid, itaconic acid-maleic acid, 1,2,3,4butanetetracarboxylic acid (BTCA), glyoxal, glycerol, 1,4 butanedioldiglycidyl ether, and a polyol.
 31. The separation media of claim 21,wherein the membrane is in the shape of a wafer, a cylinder, a sphere,or a tube.
 32. The separation media of claim 30, wherein the membrane isa hybrid cellulosic composition.
 33. A separation method comprising:flowing a fluid through the separation media of claim
 21. 34. The methodof claim 33, wherein the separation media performs a selectiveadsorption to separate molecules from the fluid.
 35. The method of claim33, further comprising recovering molecules from the separation media.36. The method of claim 33, wherein the flowing is occurring at a rateof between about 5 MV/min and about 400 MV/min.
 37. The method of claim33, wherein the flowing is occurring at a rate of between about 10MV/min and about 300 MV/min.
 38. The method of claim 33, wherein theseparation media has a dynamic binding capacity on a volume basis of atleast about 60 mg/m of the separation media.
 39. The method of claim 33,wherein the separation media has a dynamic binding capacity on a massbasis of at least about 120 mg/g of the separation media.
 40. The methodof claim 33, wherein the membrane comprises at least 40 wt. % of thecellulose.